Conventionally, accurate and precise isotope measurements are made on magnetic sector mass spectrometers, in particular magnetic sector mass spectrometers utilizing a multi-collector for simultaneous detection of isotopes. Prior to analysis, a sample typically undergoes oxidation, pyrolysis and/or reduction at an elevated temperature to produce gaseous molecules, for example one or more of COx, NOx, N2, H2O and SO2 (x=1 or 2). The gases are then introduced into the isotope ratio mass spectrometer for isotopic analysis. In the isotope ratio spectrometer, the gases are ionized and the ratios of corresponding isotopes are measured for example by comparing outputs of different collectors. The ratios of the isotopes of interest are typically measured relative to an isotopic standard in order to eliminate any bias or systematic error in the measurements.
Recently, it has been shown that electrostatic orbital trap mass spectrometers, such as an ORBITRAP mass spectrometer (Thermo Scientific), are also capable of measuring precise isotope ratios that should, in principle, be accurate as well (John Eiler, presentation at Clumped Isotope Workshop, January 2016; John Eiler et al. Poster at ASMS 2016 conference). Such results have been made using an ORBITRAP mass spectrometer employing electron impact ionization coupled to a gas chromatography (GC) column and employing a peak broadener downstream of the GC column. However, the concept of a peak broadener in the form of an in-line sweep volume is difficult to implement in an LC setup because of slower diffusion in a liquid. This would cause inhomogeneities in the effluent and therefore cause the measurement to become less reproducible.
The measurement of precise and accurate isotope ratios using liquid chromatography (LC) coupled to a mass spectrometer has presented particular problems. LC is an established technique in the field of biochemistry, life science and pharmacology for the separation of molecular components in a mixture. A typical sample includes organic molecules dissolved in an organic solvent, or an aqueous solution, or a medium comprising water and an organic solvent. For such samples, separation of the molecules from the solvent is generally carried out with an organic mobile phase using techniques such as high performance liquid chromatography (HPLC), capillary-zone electrophoresis (CZE) and size-exclusion chromatography (SEC). However, coupling an isotope ratio mass spectrometer to a liquid chromatography system presents technical challenges because the LC mobile phase is often based on organic solvent, and/or includes carbon containing buffers, and therefore produces the same species of oxidation or reduction products as organic sample molecules of interest, thus interfering with the isotopic analysis. There have been various attempts at coupling liquid chromatography to IRMS, as identified below.
“Moving-wire device for Carbon Isotopic Analyses of Nanogram Quantities of Nonvolatile Organic Carbon” (A. L. Sessions, S. P. Sylva and J. M. Hayes, Anal. Chem., 2005, 77, 6519-6527) describes a method for analyzing 13C ratios of involatile organic samples dissolved in solution. The output solution of the separation system is dried onto a nickel wire to remove the mobile phase from the sample. The residual sample is then combusted and the evolved CO2 is analyzed by IRMS. However, both the precision and sensitivity of this method are limited by a high background level of CO2 derived from carbon within the wire. The moving wire coupling has been unsuccessful commercially because of its inherent unreliability.
Another method of coupling a liquid chromatography system to an IRMS is presented in ““Continuous-Flow Isotope Ratio Mass Spectrometry Using the Chemical Reaction Interface with Either Gas or Liquid Chromatography Introduction” (Y. Teffera, J. Kusmierz, F. Abramson, Anal. Chem., 1996, 68, 1888-1894)”. In this method, the solution exiting from the liquid chromatography system undergoes desolvation at semi-permeable membranes prior to chemical oxidation of the dry aerosol. The oxidized products are then analyzed by IRMS. However, the method described does not remove the mobile phase to the required ultra-low levels of solvent, for example, to a solvent/sample ratio better than 1:100.
Wet chemical oxidation (as used by LC-Isolink™ from Thermo Fisher Scientific) allows coupling to liquid chromatography. The solution output from the chromatography system is mixed with an oxidizing agent and supplied to an oxidation reactor. In the oxidation reactor the organic compounds are converted into CO2, which is then analyzed in the IRMS. However, there is no separation of the mobile phase from the sample and, therefore, this method is not suitable for LC separation methods that utilize an organic mobile phase or liquid phase modifiers or buffers containing carbon.
In addition, problems associated with ion source fluctuations that are inherent with conventional LC-MS ion sources, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) sources, further deter the use of LC-MS for accurate and precise isotope ratio measurements.
In a recent presentation (A. Breidbach, Improved precision of measured isotope ratio through peak parking and scan-based statistics in IDMS of small organic molecules, Poster ThOS36-02, 20th International Mass Spectrometry Conference 2014, Geneva, CH), a method of peak parking has been used that involves cutting an LC peak out of a chromatogram and capturing the eluting species in a sample loop. The loop is then flushed and transferred to the mass spectrometer by a lower flow generated by a second pump. However, the steps of removing the sample from the main flow into a loop and then flushing to the mass spectrometer have not achieved the level of precision and accuracy that is often desired for isotope ratio measurements. Moreover, the system is complex, requiring the additional sample loop and second pump system.
Accordingly, there remains a need for precise and accurate isotope ratio determination using mass spectrometric detection coupled to LC.